Tumor targeting reagent and application thereof in tumor diagnosis

Abstract

The invention discloses a tumor targeting reagent and application thereof in tumor diagnosis. The targeting reagent has strong affinity to tumors, strong specificity and no biotoxicity, is relatively stable in vivo, and can be used as a molecular probe to be applied to molecular imaging and / or treatment of various integrin alpha6 high-expression tumors; the targeting reagent provided by the invention comprises the tumor targeting peptide RWYDeA, and further comprises a radionuclide chelating agent, other connecting structures and a hydroxyl substitution structure, and the structure is beneficial to improving the tumor targeting property of the targeting reagent, prolonging the tumor binding time and achieving a better tumor diagnosis effect; the method can be used for preparing products for diagnosing / treating diseases related to high expression of integrin alpha6.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a tumor-targeting reagent and its application in tumor diagnosis and treatment. Background Art

[0002] Integrin α6 is encoded by the ITGA gene and is highly expressed in thirteen types of tumors such as breast cancer, lung cancer, liver cancer, rectal adenocarcinoma, colon cancer, cholangiocarcinoma, esophageal cancer, pancreatic cancer, gastric cancer, glioma, head and neck tumors, nasopharyngeal carcinoma, and leukemia. Previously, integrin α6-targeting peptides such as CRWYDENAC, RWYD, and RWYDeA have been applied to the imaging research of some of the above-mentioned tumors and achieved good results. Based on the targeting peptide RWYDeA, this patent successfully designed a variety of tumor-targeting reagents by optimizing and adjusting its linking structure or substituting the hydroxyl groups on the amino acids of RWYDeA. Through experiments on tumor model mice and clinical trials on tumor patients, these reagents demonstrated excellent tumor-targeting performance. The successful development of these tumor-targeting reagents is of great significance for the diagnosis and treatment of tumors with high expression of integrin α6. Summary of the Invention

[0003] The purpose of the first aspect of the present invention is to provide a targeting reagent.

[0004] The purpose of the second aspect of the present invention is to provide a preparation method of the targeting reagent in the first aspect of the present invention.

[0005] The purpose of the third aspect of the present invention is to provide an application of the above-mentioned targeting reagent.

[0006] The purpose of the fourth aspect of the present invention is to provide a product containing the above-mentioned targeting reagent.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In the first aspect of the present invention, a targeting reagent is provided,

[0009] comprising an integrin α6-targeting peptide RWYDeA, and an imaging agent and / or a therapeutic agent;

[0010] The structural formula of the targeting reagent is shown as follows;

[0011]

[0012] wherein R1 is a linking structure, R2 is an imaging agent and / or a therapeutic agent; R1 exists or does not exist; R3 is a hydroxyl group, (O-SO2F), or (SO2F).

[0013] In some embodiments of the present invention, the therapeutic agent is at least one of a radionuclide, a pro-apoptotic peptide, a nanoparticle, a chemotherapeutic agent, a nanomicelle, a liposomal drug, and a cytokine.

[0014] In some embodiments of the present invention, the imaging agent is at least one of an FB group, a radionuclide, biotin, a fluorophore, a fluorescent protein, an antibody, horseradish peroxidase, and alkaline phosphatase.

[0015] In some more preferred embodiments of the present invention, the imaging agent is a radionuclide.

[0016] In some embodiments of the present invention, the radionuclide is labeled with RWYDeA through a chelating agent.

[0017] In some preferred embodiments of the present invention, the radionuclide is 18 F, 68 Ga, 64 Cu, 89 Zr, 99m Tc, 177 Lu, 131 I, 90 Y, 51 Cr, 67 Cu, 67 Ga, 111 In, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 86 Y, 149 Pm, 165 Dy, 169 Er, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 123 I, 124 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu,201 Tl, 203 Pb, 212 Pb, 198 Au, 225 Ac, 227 Th or 199 any one of Ag.

[0018] In some embodiments of the present invention, the chelating agent is any one of 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N''-pentaacetic acid (DTPA), bis-(carboxymethylimidazole) glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC).

[0019] In some embodiments of the present invention, the linking structure comprises at least one of (1) to (4):

[0020] (1) ethylene glycol (PEG) n or aspartic acid (DD) n ;

[0021] (2) (CH2) n where each -CH2- is optionally replaced by -O-, -NH-, -(CO)-, -NH-(CO)-, -CH(NH2)- or -(CO)-NH-, but no two adjacent -CH2- groups are replaced;

[0022] (3) one or more glutamic acid (E), lysine (K) or their derivative compounds;

[0023] (4) a fragment of the following structure:

[0024]

[0025] where n is an integer from 0 to 30.

[0026] In some preferred embodiments of the present invention, n is an integer from 0 to 6.

[0027] In some embodiments of the present invention, the structural formula of the targeting agent is as follows;

[0028]

[0029] In some embodiments of the present invention, the above formulas (I-1) to (I-4) can be additionally radiolabeled.

[0030] In some embodiments of the present invention, the targeting agent is a targeting agent for diseases associated with high expression of integrin α6.

[0031] In some embodiments of the present invention, the disease associated with high expression of integrin α6 is a tumor.

[0032] In some embodiments of the present invention, the tumor is breast cancer, lung cancer, liver cancer, rectal adenocarcinoma, colon cancer, cholangiocarcinoma, esophageal cancer, pancreatic cancer, gastric cancer, glioma, head and neck tumor, kidney cancer, pheochromocytoma, paraganglioma, nasopharyngeal carcinoma or leukemia.

[0033] In a second aspect of the present invention, there is provided a method for preparing the targeting agent of the first aspect of the present invention, which is synthesized using the Fmoc solid-phase synthesis strategy.

[0034] In a third aspect of the present invention, there is provided the use of the polypeptide of the first aspect of the present invention or the targeting agent of the second aspect of the present invention in the preparation of a diagnostic and / or therapeutic product for diseases associated with high expression of integrin α6.

[0035] In some embodiments of the present invention, the disease associated with high expression of integrin α6 is a tumor.

[0036] In some embodiments of the present invention, the tumor is breast cancer, lung cancer, liver cancer, rectal adenocarcinoma, colon cancer, cholangiocarcinoma, esophageal cancer, pancreatic cancer, gastric cancer, glioma, head and neck tumor, kidney cancer, pheochromocytoma, paraganglioma, nasopharyngeal carcinoma or leukemia.

[0037] In a fourth aspect of the present invention, there is provided a product comprising the polypeptide of the first aspect of the present invention or the targeting agent of the second aspect of the present invention.

[0038] In some embodiments of the present invention, the product is a diagnostic and / or therapeutic drug.

[0039] In some embodiments of the present invention, the dosage form of the drug is injection, granule, oral liquid, aerosol, capsule or spray.

[0040] In some embodiments of the present invention, the administration route of the drug is intravenous, arterial, intracavitary, subcutaneous, intramuscular, intraperitoneal, local injection or intrathecal injection.

[0041] The beneficial effects of the present invention are as follows:

[0042] The present invention provides a targeting agent comprising RWYDeA, further comprising an imaging agent and / or a therapeutic agent, which plays a better role in tumor diagnosis and treatment. The imaging agent labels the corresponding polypeptide, has better stability, and achieves a better diagnostic effect; it can be used to prepare a diagnostic / therapeutic product for diseases associated with high expression of integrin α6. Description of the Drawings

[0043] Figure 1 This is the synthetic route diagram of RWYDeA-NOTA in Example 2-1 of the present invention.

[0044] Figure 2 This is the mass spectrum diagram of RWYDeA-NOTA in Example 2-1 of the present invention

[0045] Figure 3 This is the HPLC diagram of RWYDeA-NOTA in Example 2-1 of the present invention

[0046] Figure 4 This is the synthetic route diagram of RWYDeA-OT-NOTA in Example 2-2 of the present invention.

[0047] Figure 5 This is the mass spectrum diagram of RWYDeA-OT-NOTA in Example 2-2 of the present invention.

[0048] Figure 6 This is the HPLC diagram of RWYDeA-OT-NOTA in Example 2-2 of the present invention.

[0049] Figure 7 This is the synthetic route diagram of RWYDeA-FS-NOTA in Example 2-3 of the present invention.

[0050] Figure 8 This is the mass spectrum diagram of RWYDeA-FS-NOTA in Example 2-3 of the present invention.

[0051] Figure 9 This is the HPLC diagram of RWYDeA-FS-NOTA in Example 2-3 of the present invention.

[0052] Figure 10 This is the synthetic route diagram of RWYDeA-FS-DOTA in Example 2-4 of the present invention.

[0053] Figure 11 This is the mass spectrum diagram of RWYDeA-FS-DOTA in Example 2-4 of the present invention.

[0054] Figure 12 This is the HPLC diagram of RWYDeA-FS-DOTA in Example 2-4 of the present invention.

[0055] Figure 13 In Example 4-1 of the present invention 18 F or 68 The small animal PET / CT imaging diagram of the Ga-labeled compound in the subcutaneous tumor-bearing mice of nasopharyngeal carcinoma.

[0056] Figure 14 In Example 4-2 of the present invention18 PET / CT imaging of RWYDeA-PEG4-NOTA labeled with ¹⁸F in patients with nasopharyngeal carcinoma.

[0057] Figure 15 In Example 4-2 of the present invention 18 PET / CT imaging of RWYDeA-NOTA labeled with ¹⁸F in patients with nasopharyngeal carcinoma.

[0058] Figure 16 In Example 4-2 of the present invention 18 PET / CT imaging of ¹⁸F-RWYDeA-OT-NOTA in patients with liver cancer.

[0059] Figure 17 In Example 4-2 of the present invention 18 PET / CT imaging of ¹⁸F-RWYDeA-FS-NOTA in patients with breast cancer. Detailed implementation manners

[0060] The following will clearly and completely describe the concept of the present invention and the technical effects generated in combination with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0061] Example 1 Design and synthesis of a radiopharmaceutical based on the integrin α6 targeting peptide RWYDeA

[0062] This example provides a compound that can be labeled with a radionuclide and targets integrin α6. Its structure simultaneously includes an integrin α6 targeting peptide RWYDeA and a radionuclide chelating structure; the targeting peptide and the radionuclide chelating structure are connected with or without a connecting structure. The compound includes the structure shown in the following formula (Ⅰ):

[0063]

[0064] Among them, R1 is a fragment with a connecting structure, which can be from polyethylene glycol (PEG) n or aspartic acid (DD) n short chain, or is based on (CH2) nReplacement structure of methyl, where n is an integer from 0 to 30 (preferably an integer from 0 - 6), and each -CH2- is individually replaced or not replaced with -O-, -NH-, -(CO)-, -NH-(CO)-, -CH(NH2)- or -(CO)-NH-, with the condition that no two adjacent -CH2- groups are replaced. R1 can also be with or without one or more glutamic acid (E), lysine (K) or derivative compounds containing glutamic acid and lysine, or a fragment of the following structure R1 is present or absent.

[0065] R2 is a fragment with a radionuclide chelating group selected from any one of 1,4,7,10 - tetraazacyclododecane - N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7 - triazacyclononane - 1,4,7 - triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine - N,N,N',N',N”-pentaacetic acid (DTPA), bis-(carboxymethylimidazole) glycine or 6 - hydrazinopyridine - 3 - carboxylic acid (HYNIC).

[0066] Among them, the radionuclide labeled by the R2 radionuclide chelating group is selected from 18 F, 68 Ga, 64 Cu, 89 Zr, 99m Tc, 177 Lu, 131 I, 90 Y, 51 Cr, 67 Cu, 67 Ga, 111 In, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 86 Y, 149 Pm, 165 Dy, 169 Er, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh,101m Rh, 119 Sb, 128 Ba, 123 I, 124 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 198 Au, 225 Ac, 227 Th or 199 any one of Ag.

[0067] According to the compound structural formula (I), the structure of compound RWYDeA-NOTA is preferably as formula (I-1), or the structure of compound RWYDeA-OT-NOTA is as formula (I-2).

[0068]

[0069] In addition, this example also found that when the R3 group is replaced by one of (O-SO2F) and (SO2F) from hydroxyl group, the binding time of the compound with integrin α6 is significantly prolonged. According to the compound structural formula (II), compound RWYDeA-FS-NOTA (formula II-3) and compound RWYDeA-FS-DOTA (formula II-4) are preferably selected:

[0070]

[0071] Example 2-1: Preparation of compound RWYDeA-NOTA (formula I-1)

[0072] Synthesize compound RWYDeA-NOTA according to the Fmoc solid-phase synthesis strategy, and its synthesis route is as Figure 1 ;

[0073] The synthesis process and specific parameters are as follows (taking 1 g of Wang resin as the benchmark, CAS: 1365700-43-1):

[0074] 1. Activation of the starting resin

[0075] Resin loading: The initial loading of Wang resin is usually 0.5-1.0 mmol / g (taking 0.8 mmol / g as an example);

[0076] Starting material: Fmoc-D-Glu-OAll (molecular weight is about 450 g / mol);

[0077] Feeding amount: 1.2-fold molar excess (i.e., 0.8 mmol × 1.2 = 0.96 mmol, corresponding to a mass of approximately 0.43 g);

[0078] Coupling conditions: HBTU / HOBt / DIPEA (molar ratio 1:1:2), DMF solution with a volume of 10 mL / g resin, react for 2 hours.

[0079] 2. Stepwise sequence extension

[0080] Deprotection of Fmoc: 20% piperidine / DMF solution, volume 15 mL / g resin, treated in 2 portions (5 minutes each).

[0081] Coupling step:

[0082] Amino acid feeding: Fmoc-Asp(Dde-Ethylenediamine)-OH (molecular weight approximately 600 g / mol), calculated according to 3-fold molar excess (0.8 mmol × 3 = 2.4 mmol, mass approximately 1.44 g).

[0083] Ratio of condensing agent: HBTU / HOBt / DIPEA = 1:1:2 (volume ratio), total volume of the reaction solution 20 mL / g resin.

[0084] Repeat according to the steps, sequentially couple Fmoc-Ala, Arg(Pbf), Trp(Boc), Tyr(Bu), reaction time for each step is 1 - 2 hours, wash with DMF (10 mL / g resin each time).

[0085] 3. Selective deprotection and cyclization

[0086] Removal of Dde group: 2% hydrazine hydrate / DMF solution, volume 10 mL / g resin, treated 3 times (5 minutes each).

[0087] Cyclization reaction: HATU / DIPEA (molar ratio 1:2), volume 15 mL / g resin, reaction time 24 hours. 5-fold condensing agent based on the resin loading amount (0.8 mmol × 5 = 4 mmol HATU).

[0088] 4. NOTA functionalization

[0089] Coupling of NOTA-bis(TBu) ester: NOTA ester in 2-fold molar excess (0.8 mmol × 2 = 1.6 mmol, molecular weight approximately 500 g / mol, corresponding mass 0.8 g). DCM / DMF mixture (volume ratio 1:1), total volume of the reaction solution 20 mL / g resin.

[0090] 5. Cleavage and purification

[0091] TFA cleavage: TFA / water / triisopropylsilane (volume ratio 95:2.5:2.5), dosage 20 mL / g resin, reaction for 2 hours.

[0092] Purification parameters: C18 column, mobile phase is acetonitrile / water (containing 0.1% TFA), gradient from 20% acetonitrile to 40%, flow rate 10 mL / min.

[0093] The obtained RWYDeA-NOTA has a theoretical molecular weight of 1148.2, Figure 2 as the mass spectrum of RWYDeA-NOTA, the measured molecular weight is 1148.4, and the mass spectrum result is consistent with the target compound. Figure 3 as the high performance liquid chromatography of RWYDeA-NOTA, the purity is 97.5%.

[0094] Example 2-2: Preparation of compound RWYDeA-OT-NOTA (Formula I-2)

[0095] Compound RWYDeA-OT-NOTA was synthesized according to the Fmoc solid-phase synthesis strategy, and its synthetic route is as Figure 4 ;

[0096] The synthesis process and specific parameters are as follows (based on 1 g of Wang resin):

[0097] 1. Activation of starting resin

[0098] Resin loading: The initial loading of Wang resin is 0.8 mmol / g;

[0099] Starting material: Fmoc-D-Glu-OAll (molecular weight about 450 g / mol);

[0100] Dosage: 1.2-fold molar excess (i.e., 0.8 mmol × 1.2 = 0.96 mmol, corresponding to a mass of about 0.43 g);

[0101] Coupling conditions: HBTU / HOBt / DIPEA (molar ratio 1:1:2), a DMF solution with a volume of 10 mL / g resin, reaction for 2 hours.

[0102] 2. Stepwise extension of the sequence

[0103] Removal of Fmoc protection: 20% piperidine / DMF solution, volume 15 mL / g resin, treated in 2 times (5 minutes each time).

[0104] Coupling step:

[0105] Amino acid feeding: Fmoc-Asp(Dde-Ethylenediamine)-OH (molecular weight about 600 g / mol), calculated by 3-fold molar excess (0.8 mmol × 3 = 2.4 mmol, mass about 1.44 g).

[0106] Ratio of condensing agent: HBTU / HOBt / DIPEA = 1:1:2 (volume ratio), total volume of reaction solution 20 mL / g resin.

[0107] Repeat according to the steps, sequentially couple Fmoc-Ala, Arg(Pbf), Trp(Boc), Tyr(Bu), reaction time for each step is 1 - 2 hours, wash with DMF (10 mL / g resin each time).

[0108] 3. Removal of Dde group: 2% hydrazine hydrate / DMF solution, volume 10 mL / g resin, treat for 3 times (5 minutes each time).

[0109] Cyclization reaction: HATU / DIPEA (molar ratio 1:2), volume 15 mL / g resin, reaction time 24 hours. 5-fold condensing agent of resin loading amount (0.8 mmol × 5 = 4 mmol HATU).

[0110] 4. OT-NOTA functionalization

[0111] Coupling of NOTA-bis(tBu)ester-Glu(Oxythiamine): NOTA-bis(tBu)ester by 2-fold molar excess (1.6 mmol), DCM / DMF mixed solution (volume ratio 1:1), total volume of reaction solution 20 mL / g resin.

[0112] 5. Cleavage and purification

[0113] TFA / water / triisopropylsilane = 95:2.5:2.5 (20 mL / g resin, total 19 mL TFA + 0.5 mL H2O + 0.5 mL TIS), react for 2 hours, stir in ice bath.

[0114] C18 reversed-phase column, gradient elution with acetonitrile / water (containing 0.1% TFA) (20% → 40% acetonitrile), flow rate 10 mL / min.

[0115] The theoretical molecular weight of the obtained RWYDeA-OT-NOTA is 1525.7, Figure 5 is the mass spectrum of RWYDeA-OT-NOTA, the measured molecular weight is 1525, and the mass spectrum result is consistent with the target substance. Figure 6 is the high performance liquid chromatography of RWYDeA-OT-NOTA, and the purity is 90.0%.

[0116] Example 2-3: Preparation of Compound RWYDeA-FS-NOTA (Formula I-3)

[0117] Compound RWYDeA-FS-NOTA was synthesized according to the Fmoc solid-phase synthesis strategy, and its synthetic route is as Figure 7 ;

[0118] The synthesis process and specific parameters are as follows (based on 1 g of Wang resin):

[0119] 1. Activation of the starting resin: The same as in Example 2.1.

[0120] 2. Deprotection of the Fmoc protecting group

[0121] The 20% piperidine / DMF solution was treated twice (5 minutes each time) at 15 mL / g resin, and washed with DMF three times (10 mL / g resin each time).

[0122] 3. Coupling of the Asp(Dde-NOTA) fragment

[0123] Amino acid: Fmoc-Asp(Dde-NOTA-bis(t-Bu ester)-Ethylenediamine)-OH (molecular weight ≈ 800 g / mol), 3-fold molar excess (0.8 mmol × 3 = 2.4 mmol), HBTU / HOBt / DIPEA (molar ratio 1:1:2), volume 20 mL / g resin, react for 2 hours under helium protection.

[0124] 4. Stepwise coupling of Ala, Arg(Pbf), Trp(Boc), Tyr(O-SO2F)

[0125] The amino acid feed was in 3-fold molar excess, the solvent volume was 15 mL DMF / g resin, and the condensing agent HBTU / DIPEA = 1:2 (molar ratio).

[0126] Key protecting groups: Arg(Pbf), Trp(Boc), Tyr(O-SO2F) need to be retained throughout until cleavage.

[0127] 5. Selective deprotection and cyclization

[0128] Deprotection of Dde: 2% hydrazine hydrate / DMF solution, 10 mL / g resin, treated 3 times (5 minutes each time).

[0129] Deprotection of OAll: Catalyzed by Pd(PPh3)4 (0.1 mmol / g resin), solvent: treated with DCM / anisole (volume 5 mL / g resin).

[0130] Cyclization reaction: Condensing agent: HATU / DIPEA (5-fold excess, 0.8 × 5 = 4 mmol HATU, mass ≈ 1.52 g). Solvent: 15 mL / g resin (15 mL DMF, reacting for 24 hours under nitrogen protection).

[0131] 6. TFA cleavage and purification

[0132] TFA / water / triisopropylsilane = 95:2.5:2.5 (20 mL / g resin, a total of 19 mL TFA + 0. mL H2O + 0.5 mL TIS), reacting for 2 hours, stirring in an ice bath.

[0133] C18 reverse-phase column, gradient elution with acetonitrile / water (containing 0.1% TFA) (20% → 40% acetonitrile), flow rate 10 mL / min.

[0134] The theoretical molecular weight of the obtained RWYDeA-FS-NOTA is 1230.3, Figure 8 is the mass spectrum of RWYDeA-FS-NOTA, the measured molecular weight is 1230.4, and the mass spectrum result is consistent with the target substance. Figure 9 is the high-performance liquid chromatography diagram of RWYDeA-FS-NOTA, and the purity is 96.5%.

[0135] Example 2 - 4: Preparation of compound RWYDeA-FS-DOTA (Formula I - 4)

[0136] Synthesize compound RWYDeA-FS-DOTA according to the Fmoc solid-phase synthesis strategy, and its synthesis route is as Figure 10 ;

[0137] The synthesis process and specific parameters are as follows (based on 1 g of Wang resin):

[0138] 1. Activation of the starting resin: The same as in Example 2.1.

[0139] 2. Removal of the Fmoc protecting group:

[0140] 20% piperidine / DMF solution is treated 2 times according to 15 mL / g resin (5 minutes each time), and washed 3 times with DMF (10 mL / g resin each time).

[0141] 3. Coupling of the Asp(Dde-Ethylenediamine) fragment

[0142] Amino acid Fmoc-Asp(Dde-Ethylenediamine)-OH (molecular weight ≈ 600 g / mol) is in 3-fold molar excess, condensing agent HBTU / DIPEA (molar ratio 1:2), volume 20 mL / g resin.

[0143] 4. Couple Ala, Arg(Pbf), Trp(Boc), and Tyr(O-SO2F) step by step

[0144] The amino acid feed is in a 3-fold molar excess, the solvent volume is 15 mL DMF / g resin, and the condensing agent HBTU / DIPEA = 1:2 (molar ratio).

[0145] Key protecting groups: Arg(Pbf), Trp(Boc), and Tyr(O-SO2F) must be retained throughout until cleavage.

[0146] 5. Selective deprotection and cyclization

[0147] Remove the Dde protection: 2% hydrazine hydrate / DMF solution, 10 mL / g resin, treat 3 times (5 minutes each).

[0148] Remove the OAll protection: Catalyzed by Pd(PPh3)4 (0.1 mmol / g resin), solvent: DCM / anisole (volume 5 mL / g resin) treatment.

[0149] Cyclization reaction: Condensing agent: HATU / DIPEA (5-fold excess, 0.8×5 = 4 mmol HATU, mass ≈ 1.52 g). Solvent: 15 mL / g resin (15 mL DMF, react for 24 hours under nitrogen protection).

[0150] 6. Coupling of DOTA(tBu)3

[0151] DOTA(tBu)3 (molecular weight ≈ 800 g / mol) is in a 2-fold molar excess, and coupling is carried out with DCM / DMF (1:1, volume 20 mL / g resin).

[0152] 7. TFA cleavage and purification

[0153] TFA / water / triisopropylsilane (volume ratio 95:2.5:2.5) (20 mL / g resin, a total of 19 mL TFA + 0.5 mL H2O + 0.5 mL TIS), react for 2 hours, stir in an ice bath.

[0154] Precipitation and washing: Precipitate with cold ether, wash with ether 3 times (20 mL each) after centrifugation.

[0155] C18 reversed-phase column, gradient elution with acetonitrile / water (containing 0.1% TFA) (20% → 40% acetonitrile), flow rate 10 mL / min.

[0156] The theoretical molecular weight of the obtained RWYDeA-FS-DOTA is 1331.4, Figure 11 which is the mass spectrum of RWYDeA-FS-DOTA. The measured molecular weight is 1331.4, and the mass spectrum result is consistent with the target substance. Figure 12This is the high performance liquid chromatography (HPLC) chromatogram of RWYDeA-FS-DOTA, with a purity of 98.7%.

[0157] Example 3-1: Preparation of Radioactive 18 F-Labeled Compound

[0158] Take 50 μg of the compound (suitable for compounds containing NOTA), 6.275 μg of AlCl3·6H20, 330 μL of absolute ethanol, 65 μL of 18 F water with a radioactivity of about 185 - 1850 MBq, and 4 - 5 μL of 10% glacial acetic acid to adjust the pH of the reaction solution to about 3.8 - 4.2. Mix well and place in a 10 mL vacuum glass bottle. Heat in a 100 °C metal bath for 10 minutes and let stand at room temperature for 5 minutes. Activate the Waters Sep-Pak C18 column, wash with 10 mL of absolute ethanol, and then wash with 10 mL of sterile water. Add 10 mL of sterile water to the glass bottle, mix well, transfer to the activated C18 column, and wash with 20 mL of sterile water. Elute with 400 μL of 70% ethanol water. Dilute with normal saline and filter through a sterile filter to obtain 18 F-Labeled Compound Injection

[0159] Example 3-2: Preparation of Radioactive 68 Ga-Labeled Compound

[0160] Take 50 μg of the compound (suitable for compounds containing NOTA and DOTA), 1 mL of 68 Ga ion water with a radioactivity of about 185 - 1850 MBq, and 0.8 - 1 mL of 0.15 M sodium acetate (NaAc) buffer solution (pH = 7.2) to adjust the pH of the reaction solution to about 3.5 - 4. Mix well and place in a 10 mL vacuum glass bottle. Heat in a 40 - 100 °C metal bath for 15 minutes and let stand at room temperature for 5 minutes. Dilute with normal saline and filter through a sterile filter to obtain 68 Ga-Labeled Compound Injection

[0161] Example 4-1: 18 F or 68 PET / CT Imaging of Ga-Labeled Compounds in Mice with Nasopharyngeal Carcinoma Subcutaneous Tumors

[0162] 1. Use 18 F-RWYDeA-PEG4-NOTA (a previously reported molecule, used as a negative control, https: / / doi.org / 10.1016 / j.bioorg.2024.107892), 18 F-RWYDeA-NOTA, 18 F-RWYDeA-OT-NOTA, 18F-RWYDeA-FS-NOTA or 68 Ga-RWYDeA-FS-DOTA was injected into the subcutaneous tumor-bearing mice via the tail vein, and small animal PET / CT imaging was performed 3 hours after the injection.

[0163] The results are as Figure 13 shown, 18 F-RWYDeA-PEG4-NOTA, 18 F-RWYDeA-NOTA, 18 F-RWYDeA-OT-NOTA, 18 F-RWYDeA-FS-NOTA or 68 Ga-RWYDeA-FS-DOTA showed high uptake in the kidneys and bladder excretion organs of the subcutaneous tumor-bearing mice with nasopharyngeal carcinoma, and low uptake in other organs, which are the basic characteristics of polypeptide drugs. 18 F-RWYDeA-PEG4-NOTA had a small amount of uptake in the tumor, and its maximum standardized uptake value SUV max was 2.51. Relatively, the four compounds of this patent 18 F-RWYDeA-NOTA, 18 F-RWYDeA-OT-NOTA, 18 F-RWYDeA-FS-NOTA or 68 Ga-RWYDeA-FS-DOTA showed a significant increase in uptake in the tumor, and the SUV max was 4.55, 8.42, 10.03 and 5.70 respectively, with an increase of 81.27%, 235.46%, 299.60% and 127.92% respectively, and the tumor targeting effect was significantly improved.

[0164] Example 4-2: 18 PET / CT imaging of F-labeled compounds in tumor patients

[0165] The investigator-initiated IIT clinical trial was approved by the Clinical Research Ethics Committee of the Affiliated Hospital of Jiangnan University. According to the patient's weight, the patient was intravenously injected with a dose of about 314.5 MBq and a volume of about 8 mL of 18 F-labeled compound. After 1 hour, whole-body static PET / CT scanning was performed, and whole-body static FDG-PET / CT scanning was performed the next day for comparison. The SUV was automatically calculated by drawing regions of interest on the axial images. max .

[0166] Patient A was diagnosed with nasopharyngeal carcinoma after imaging and pathological diagnosis, and was injected with 314.5 MBq of 18 F-RWYDeA-PEG4-NOTA. The results are as Figure 14 shown.18 F-FDG occupied the left top and left walls of the nasopharynx, and the metabolism was abnormally increased, which was consistent with the signs of malignant lesions. Combined with the imaging results, it was determined that 18 The areas with high F-FDG uptake are nasopharyngeal carcinoma lesions. 18 F-RWYDeA-PEG4-NOTA had no uptake or low uptake in the same lesion site, which means that its imaging effect on NPC was poor.

[0167] Patient B was diagnosed with nasopharyngeal carcinoma with skull base invasion after imaging and pathological diagnosis. The injection dose was 314.5 MBq. 18 F-RWYDeA-NOTA. The result is Figure 15 As shown, 18 F-RWYDeA-NOTA has strong uptake in the nasopharynx near the skull base. The strong signal at the uptake location has a clear boundary with the surrounding tissues. The uptake location is consistent with the lesion location suggested by the head and neck MRI. max The value is 3.8. 18 F-FDG has vigorous sugar metabolism in the brain, which leads to a high imaging background and affects the judgment of head lesions. No abnormal high uptake points were found in the nasopharynx near the skull base, and the specificity was slightly lower. 18 F-RWYDeA-NOTA has a better tumor diagnostic effect for patients with nasopharyngeal carcinoma, especially those with skull base invasion.

[0168] Patient C was diagnosed with liver cancer after imaging and pathological diagnosis, and the injection dose was 314.5MBq 18 F-RWYDeA-OT-NOTA. The result is Figure 16 As shown, 18 The high uptake position of F-RWYDeA-OT-NOTA is consistent with the position of the tumor indicated by the CT shadow, i.e. 18 F-RWYDeA-OT-NOTA was significantly taken up in liver tumors, and SUV max The value is 4.2, and it can image cancer thrombi, with a low background of normal liver tissue. 18 F-FDG can only image large tumor lesions, but not small tumors and cancer thrombi, and its sensitivity is slightly lower. 18 F-RWYDeA-OT-NOTA has a better tumor diagnostic effect on liver cancer, especially tiny lesions and cancer thrombi.

[0169] Patient D was diagnosed with breast cancer after imaging and pathological diagnosis, and the injection dose was 314.5MBq 18 F-RWYDeA-FS-NOTA. The result is Figure 17 As shown, 18 F-RWYDeA-FS-NOTA high uptake position with 18The location of the tumor indicated by F-FDG was consistent, that is 18 F-RWYDeA-FS-NOTA showed significant uptake in breast cancer tumors and low background in normal muscle tissue, with an SUV max value of 2.8, slightly higher than 18 that of F-FDG at 2.7. 18 F-RWYDeA-FS-NOTA had excellent imaging effect on breast cancer.

[0170] Based on the PET / CT imaging results of the small animals in Example 4-1 and the patients in Example 4-2, compared with the publicly disclosed compound RWYDeA-PEG4-NOTA, the four compounds RWYDeA-NOTA, RWYDeA-OT-NOTA, RWYDeA-FS-NOTA, and RWYDeA-FS-DOTA in this patent had stronger tumor targeting ability, excellent tumor imaging effect in patients, and greater clinical application value.

[0171] The above specific embodiments have described the present invention in detail, but the present invention is not limited to the above embodiments. Various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the relevant art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A targeting agent, characterized in that, it comprises an integrin α6 targeting peptide RWYDeA, and an imaging agent and / or a therapeutic agent; the structural formula of the targeting agent is as follows; wherein R1 is a linking structure, R2 is an imaging agent and / or a therapeutic agent; R1 may or may not exist; R3 is a hydroxyl group, (O-SO2F) or (SO2F).

2. The targeting agent according to claim 1, characterized in that: the therapeutic agent is at least one of a radionuclide, a pro-apoptotic peptide, a nanoparticle, a chemotherapeutic agent, a nanodroplet, a liposomal drug and a cytokine; the imaging agent is at least one of an FB group, a radionuclide, biotin, a fluorophore, a fluorescent protein, an antibody, horseradish peroxidase and alkaline phosphatase.

3. The targeting agent according to claim 1, characterized in that, the imaging agent is at least one of an FB group, a radionuclide, biotin, a fluorophore, a fluorescent protein, an antibody, horseradish peroxidase and alkaline phosphatase; preferably, the imaging agent is a radionuclide; preferably, the radionuclide is linked to RWYDeA through a chelating agent; Preferably, the radionuclide is 18 F, 68 Ga, 64 Cu, 89 Zr, 99m Tc, 177 Lu, 131 I, 90 Y, 51 Cr, 67 Cu, 67 Ga, 111 In, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 86 Y, 149 Pm, 165 Dy, 169 Er, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 123 I, 124 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 198 Au, 225 Ac, 227 Th or 199 Ag; preferably, the chelating agent is any one of 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N”-pentaacetic acid (DTPA), bis-(carboxymethylimidazole) glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC).

4. The targeting agent according to claim 1, characterized in that: the linking structure R1 comprises at least one of (1) to (4): (1) Ethylene glycol (PEG) n or aspartic acid (DD) n ; (2)(CH2) n , where each -CH2- is replaced with or without -O-, -NH-, -(CO)-, -NH-(CO)-, -CH(NH2)- or -(CO)-NH-, provided that no two adjacent -CH2- groups are replaced; (3) one or more glutamic acids (E), lysines (K) or their derivative compounds; (4) a fragment of the following structure: where n is an integer from 0 to 30.

5. The targeting reagent according to claim 4, wherein The structural formula of the targeting agent is as follows:

6. The preparation method of the targeting agent according to any one of claims 1 to 5 is obtained by using the Fmoc solid-phase synthesis strategy.

7. The application of the targeting agent according to any one of claims 1 to 5 in the preparation of a diagnostic and / or therapeutic product for integrin α6 overexpression-related diseases.

8. The application according to claim 7, characterized in that, The integrin α6 overexpression-related diseases are tumors.

9. The application according to claim 8, characterized in that: the tumors are breast cancer, lung cancer, liver cancer, rectal adenocarcinoma, colon cancer, cholangiocarcinoma, esophageal cancer, pancreatic cancer, gastric cancer, glioma, head and neck tumors, kidney cancer, pheochromocytoma, paraganglioma, nasopharyngeal carcinoma or leukemia.

10. A product, the product contains the targeting agent according to any one of claims 1 to 5; preferably, the product is a diagnostic and / or therapeutic drug; preferably, the dosage form of the drug is an injection, a granule, an oral liquid, an aerosol, a capsule or a spray; preferably, the administration route of the drug is intravenous, arterial, intracavitary, subcutaneous, intramuscular, peritoneal, local injection or intrathecal injection.

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